Abstract-P450-dependent arachidonic acid (AA) metabolites regulate arterial tone by modulating calcium-activated (BK) potassium channels in vascular smooth muscle cells (VSMC). Because eicosapentaenoic acid (EPA) has been reported to improve vascular function, we tested the hypothesis that P450-dependent epoxygenation of EPA produces alternative vasoactive compounds. We synthesized the 5 regioisomeric epoxyeicosattrienoic acids (EETeTr) and examined them for effects on K ϩ currents in rat cerebral artery VSMCs with the patch-clamp technique. 11(R),12(S)-epoxyeicosatrienoic acid (50 nmol/L) was used for comparison and stimulated K ϩ currents 6-fold at ϩ60 mV. However, 17(R),18(S)-EETeTr elicited a more than 14-fold increase. 17(S),18(R)-EET and the remaining four regioisomers were inactive. The effect of 17(R),18(S)-EETeTr was blocked by tetraethylammonium but not by 4-aminopyridine. VSMCs expressed P450s 4A1 and 4A3. Recombinant P450 4A1 hydroxylated EPA at C-19 and C-20 and epoxygenated the 17,18-double bond, yielding the R, S-and S, R-enantiomers in a ratio of 64:36. We conclude that 17(R),18(S)-EETeTr represents a novel, potent activator of BK potassium channels. Furthermore, this metabolite can be directly produced in VSMCs. We suggest that 17(R),18(S)-EETeTr may function as an important hyperpolarizing factor, particularly with EPA-rich diets. Key Words: vascular smooth muscle cells Ⅲ endothelium-derived factors Ⅲ potassium channels Ⅲ cytochrome P450 D ietary fish oil or purified (n-3) long-chain polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) exert a wide range of beneficial effects on vascular function. 1,2 Endothelium-dependent relaxation is enhanced and the vasoconstrictor response to angiotensin II and norepinephrine is reduced because of mechanisms that are incompletely understood. [3][4][5][6] Possibly, EPA and other (n-3) PUFA compete with arachidonic acid (AA) for enzymatic conversion by P450 enzymes. This competition may lead to a reduced formation of vasoactive AA metabolites while alternative metabolites originating from EPA are increased. The P450-dependent AA metabolites result from epoxygenation and hydroxylation and include the epoxyeicosatrienoic acids (EET) 5,6-, 8,9-, 11,12 to 14,15-EET, and the /(-1)-hydroxyeicosatetraenoic acids . 7 EETs are produced in the endothelium by the P450 subfamilies 2C and 2J. 8,9 EETs activate large-conductance, calcium-activated (BK) K ϩ channels in vascular smooth muscle cells (VSMC) and are considered as leading candidates for the endothelium-derived hyperpolarizing factor (EDHF). 8,10,11 20-HETE is produced by P450 4A enzymes in VSMC and acts as endogenous vasoconstrictor that inhibits BK channels. 20-HETE is important for the autoregulation of renal and cerebral blood flow. [12][13][14][15] How AA metabolite production is influenced by EPA competition for P450 enzymes, and whether or not P450-dependent EPA metabolites modulate BK channels, is unknown. We synthesized the five possible regioisomeric epoxyeicosatetraenoic acids: 5,6...